Conversion of somatic cells into functional spinal motor neurons, and methods and uses thereof

a somatic cell and motor neuron technology, applied in the field of somatic cell transdifferentiation, can solve the problems of not being able to not being able to routinely isolate analogous populations of human neurons or fully study differentiated central neurons, and not being able to refine subtype specific properties and other issues, to achieve the effect of increasing protein expression of brn2, increasing protein expression of myt1l, and increasing protein expression of isl1

Active Publication Date: 2015-01-22
CHILDRENS MEDICAL CENT CORP +1
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Benefits of technology

[0018]In some embodiments, in increase in the protein expression of least three MN-inducing factors selected from any of: Lhx3, Ascl1, Brn2, Myt1l, Isl1, Hb9, Ngn2 or NeuroD1 can be achieved by contacting a somatic cell, e.g., a fibroblast with an agent which increases the expression of the MN-inducing factor, where an agent can be selected from the group consisting of: a nucleotide sequence, a nucleic acid analogue (e.g., Locked nucleic acid (LNA), modified RNA (modRNA)), a protein, an aptamer and small molecule, ribosome, RNAi agent and peptide-nucleic acid (PNA) and analogues or variants thereof. In some embodiments, protein expression is increased by introducing at least three nucleic acid sequences encoding at least three MN-inducing factors selected from any of: Lhx3, Ascl1, Brn2, Myt1l, Isl1, Hb9, Ngn2 or NeuroD1, or encoding a functional fragment thereof, in the somatic cell, e.g., fibroblast.
[0026]In some embodiments, protein expression of NeuroD1 is increased by introducing a nucleic acid sequence encoding a NeuroD1 polypeptide comprising SEQ ID NO: 15 or SEQ ID NO: 16 or a functional fragment of SEQ ID NO: 15 or SEQ ID NO: 16 into the somatic cell, e.g., fibroblast.

Problems solved by technology

However, their individual qualities also render particular neurons either resistant or sensitive to particular degenerative stimuli.
However, it is not routinely possible to isolate analogous populations of human neurons or to isolate and fully study differentiated central neurons.
Furthermore, in many cases the neuronal populations produced from stem cells have not been shown to possess refined subtype specific properties and may only superficially resemble their counterparts from the CNS (Peljto and Wichterle, 2011).
However, it is unclear whether these cells are molecularly and functionally equivalent to embryo- or ESC-derived dopaminergic neurons.
In particular, it has yet to be determined whether any type of neuron made by reprogramming can survive and properly integrate into the CNS.

Method used

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  • Conversion of somatic cells into functional spinal motor neurons, and methods and uses thereof
  • Conversion of somatic cells into functional spinal motor neurons, and methods and uses thereof
  • Conversion of somatic cells into functional spinal motor neurons, and methods and uses thereof

Examples

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example 1

[0324]In order to determine whether transcription factors can bestow a precise neural subtype identity, we sought factors that could reprogram fibroblasts into spinal motor neurons. Here, the inventors demonstrate induction of motor neurons based on their significant translational utility and because the developmental origins and functional properties of this neural subtype are among the most well understood.

[0325]The inventors demonstrate that when mouse fibroblasts express factors previously found to induce reprogramming toward a generic neuronal phenotype (Vierbuchen et al., 2010), they also respond to components of the transcription factor network that act in the embryo to confer a motor neuron identity on committed neural progenitors. Thus, the inventors demonstrated that forced expression of these transcription factors converted mouse fibroblasts into induced motor neurons (iMNs).

[0326]Importantly, the inventors demonstrated that the resulting iMNs had a gene expression progra...

example 2

11 Factors Convert Fibroblasts into Hb9::GFP+ Cells with Neuronal Morphologies

[0327]The inventors assessed if transcription factors known to instruct motor neuron formation during development might also facilitate the conversion of other cell types into motor neurons. The inventors used the literature to select eight candidate transcription factors that participate in varied stages of motor neuron specification (Jessell, 2000). In order to potentially aid the transition toward a neuronal phenotype, the inventors supplemented the motor neuron specification factors with three factors that convert fibroblasts into induced neurons (iNs) of a generic character (Ascl1, Brn2 and Myt1l) (Vierbuchen et al., 2010) (FIG. 1A).

[0328]For reprogramming studies, mouse embryonic fibroblasts (MEFs) were harvested from Hb9::GFP mouse embryos at day E12.5, allowing spinal motor neuron conversion to be monitored. Prior to use, cultures of MEFs were carefully screened for the absence of any contaminating...

example 3

iMNs are Efficiently Induced by 7 Factors

[0330]To determine which of the 11 factors were necessary for generating iMNs, each gene was omitted one at a time (FIG. 8B). Excluding either Lhx3 or Ascl1 eliminated iMN formation. However, reprogramming efficiency was either only slightly reduced or unchanged when each of the remaining factors were removed (FIG. 8B). Interestingly, ectopic expression of Hb9 was not required for iMN formation (FIG. 8B), demonstrating that, at least in that case, exogenous Hb9 was not simply transactivating its own promoter. Similarly, Isl1 / 2 expression was detected by immunostaining in iMNs (80.6%, n=36), even when the Isl1 retrovirus was omitted from the transduction (FIGS. 1C-D and FIG. 8B).

[0331]Although Lhx3 and Ascl1 seemed necessary for reprogramming, they were not sufficient to induce motor neuron formation (FIG. 8C). However, when Lhx3 was combined with the three iN factors (Ascl1, Brn2 and Myt1l), a modest number of Hb9:: GFP+ cells were detected (...

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Abstract

The present invention provides methods of transdifferentiation of somatic cells, for example, directly converting a somatic cell of a first cell type, e.g., a fibroblast into a somatic cell of a second cell type, are described herein. In particular, the present invention generally relates to methods for converting a somatic cell, e.g., a fibroblast into a motor neuron, e.g., an induced motor neuron (iMN) with characteristics of a typical motor neuron. The present invention also relates to an isolated population comprising induced motor neurons (iMNs), compositions and their use in the treatment of motor neuron diseases such as ALS and SMA. In particular, the present invention relates to direct conversion of a somatic cell to an induced motor neuron (iMN) having motor neuron characteristics by increasing the protein expression of at least three motor-neuron inducing (MN-inducing) factors selected from Lhx3, Ascl1, Brn2, Myt1l, Isl1, Hb9, Ngn2 or NeuroD1 in a somatic cell, e.g., a fibroblast to convert the fibroblast to an induced motor neuron (iMN) which exhibits at least two characteristics of an endogenous motor neuron.

Description

CROSS REFERENCE TO RELATED APPLICATION[0001]This application claims benefit under 35 U.S.C. §119(e) of the U.S. Provisional Application Ser. No. 61 / 524,599 filed on Aug. 17, 2011, the contents of which is incorporated herein in its entirety by reference.GOVERNMENT SUPPORT[0002]This invention was made with made with Government Support under Contract Nos: NS069395, HD045732, and GM007592 awarded by the National Institutes of Health. The Government has certain rights in the invention.FIELD OF INVENTION[0003]The invention relates to methods for transdifferentiation of a somatic cell, e.g., a fibroblast to a cell having motor neuron characteristics. The present invention also relates to an isolated population comprising induced motor neurons, compositions and their use in the treatment of motor neuron diseases and disorders (MNDs).SEQUENCE LISTING[0004]The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by re...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/30
CPCA61K35/30C12N2506/1307C12N2506/13C12N2510/00C12N2501/60C12N5/0619A61K48/00Y02A50/30
Inventor EGGAN, KEVIN CARLWOOLF, CLIFFORD J.WAINGER, BRIAN J.ICHIDA, JUSTIN K.SON, ESTHER YESDE
Owner CHILDRENS MEDICAL CENT CORP
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